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Effect of intrauterine HIV-1 exposure on the frequency and function of uninfected newborns’ dendritic cells

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Effect of intrauterine HIV-1 exposure on the frequency and function of uninfected newborns’ dendritic cells
  See discussions, stats, and author profiles for this publication at: Effect of intrauterine HIV-1 exposure on thefrequency and function of uninfectednewborns' dendritic cells  Article   in  Clinical Immunology · April 2008 DOI: 10.1016/j.clim.2007.11.004 · Source: PubMed CITATIONS 24 READS 29 6 authors , including:Claire ChougnetCincinnati Children's Hospital Medical Center 159   PUBLICATIONS   4,126   CITATIONS   SEE PROFILE All content following this page was uploaded by Claire Chougnet on 28 November 2014.The user has requested enhancement of the downloaded file. All in-text references underlined in blueare linked to publications on ResearchGate, letting you access and read them immediately.  This article was published in an Elsevier journal. The attached copyis furnished to the author for non-commercial research andeducation use, including for instruction at the author’s institution,sharing with colleagues and providing to institution administration.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Effect of intrauterine HIV-1 exposure on thefrequency and function of uninfected newborns ’ dendritic cells Paula A. Velilla a,b,c , Carlos J. Montoya a , Alvaro Hoyos a , Maria E. Moreno a ,Claire Chougnet b,c,1 , Maria T. Rugeles a, ⁎ ,1 a Group of Immunovirology, School of Medicine, University of Antioquia, Medellin, Colombia b Division of Molecular Immunology, Cincinnati Children ’ s Hospital Research Foundation, Cincinnati, OH, USA c Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA Received 30 August 2007; accepted with revision 10 November 2007Available online 16 January 2008 Abstract  Immaturity of the neonatal immune system is considered an underlying factor forenhanced severity of infections in newborns. Functional defects of neonatal antigen-presentingcellsleadtodefectiveT-cellresponses.Tcellsfromuninfectedneonatesexposed inutero toHIV-1(EU) exhibit phenotypic and functional alterations; however, the function of their circulatingdendriticcells(DCs)hasnotbeencharacterized.WehypothesizedthatanHIV-1-infectedmaternalenvironmentmayinfluencetheinfants ’ DCnumber,phenotypeandfunction.EUexhibitedahigherpercentage of myeloid DCs (mDCs) than unexposed neonates, although this frequency remainedlower than that observed in adults. Plasmacytoid DC (pDC) frequencies were similar in all groups,although both groups of infants tended to have lower frequencies than adults. After LPSstimulation, mDCs from EU up-regulated CD80, CD86 and B7-H1, whereas mDCs from unexposedinfants upregulated B7-H1, but not CD80/CD86, and adult mDCs up-regulated mainly CD80 andCD86. IFN- α  production was similar in all groups, indicating a normal pDC function. Therefore,  inutero exposuretoHIV-1inducesquantitativeandqualitativechangesinneonatalDCs,particularlyin mDCs, which might be associated with alterations observed in Tcells from these EU.© 2007 Elsevier Inc. All rights reserved. KEYWORDS HIV-1;Neonates;Dendritic cells;CD80;CD86;B7-H1;IFN- α ;CpG ODN;LPS  Abbreviations:  APCs, Antigen-presenting cells; CB, cord blood; CBMC, cord blood mononuclear cell; CpG ODN, oligodeoxyribonucelotideswith CpG motifs; DC, dendritic cells; EU, HIV-1-uninfected neonates born to HIV-1-infected mothers; MC, mononuclear cell; mDC, myeloiddendritic cell; MFI, mean fluorescence intensity; pDC, plasmacytoid dendritic cell; PB, peripheral blood; UN, unexposed neonates (born toHIV-1 negative mothers). ⁎  Corresponding author. Group of Immunovirology, University of Antioquia, Calle 62, No. 52-29, Torre 2, Lab 532, Medellin, Colombia. Fax:+57 4 210 64 81. E-mail address: (M.T. Rugeles). 1 C.C. and M.T.R. contributed equally to this paper.1521-6616/$  –  see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.clim.2007.11.004 available at Clinical Immunology (2008)  126 , 243 – 250  Author's personal copy Introduction The high rate of mortality from infections in newborns islikely to arise as a consequence of the immaturity of theimmune system, particularly a functional impairment of neonatal Tcells has been described [1,2]. However, the factthat neonatal T cells show a normal response to TCR-independent signals and that defective TCR-dependentstimulation is restored by additional costimulation suggestthat an impairment of neonatal Tcell function is not only theconsequence of an intrinsic deficiency of neonatal Tcells butmay also result from inadequate signals delivered byneonatal antigen-presenting cells (APCs).Several studies suggest that neonatal APCs are function-ally altered. The main dysfunction is detected at the level of costimulatory molecule expression (reviewed in Velilla et al.[3]). Most studies have focused on the evaluation of thecostimulatory molecules CD80 and CD86 in neonatal mono-cyte-derived dendritic cells (MDDCs), a model of mDCs. Sofar, the expression of inhibitory molecules on neonatal DCsand their role in the functional alterations of these DCs hasnot been reported; in particular, B7-H1 expression has notyet been studied. B7-H1 is a recently identified moleculebelonging to the B7 family, expressed by DCs (reviewed in[4]). It inhibits Tcell function in different models [5,6]. Cytokine production by cord blood (CB) andneonatal APCsis also decreased, particularly IL-12 and IL-15 [7,8]. Plasmacytoid DCs are the main producers of Type I IFN(reviewed in [9]), and contradictory data have been published concerning the capacity of neonatal DCs toproduce IFN- α  [10 – 12].Part of the vertically transmitted HIV-1 infection takesplace  in utero , indicating that the placental barrier ispermeable to maternal HIV-1. In addition, the fetus isexposed to circulating viral proteins such as gp120. In fact, aproportion of HIV-1-uninfected neonates born to HIV-1-infected mothers (EU) exhibit HIV-1-specific T cells [13,14].Interestingly, leukocytes, particularly T cells, of EU arefunctionally altered [15,16], with some of these abnormal-ities persisting over time [16,17]. These findings suggest thatHIV-1 or its products are present in the maternal environ-ment and that, even in the absence of infection, they canalter the development of the immune system in the EU.Despite the fact that DCs play a critical role by providingthe signals required to induce immune responses [18], theirfrequency, phenotype and functional activity have not beenevaluated in EU. Therefore, we studied whether intrauterineexposure to HIV-1 influences the newborns ’  and infants ’  DCsnumber, phenotype and function, particularly the expressionof the costimulatory molecules CD80 and CD86, as well as of the inhibitory molecule B7-H1. Materials and methods Study population Thisprospectiveobservationalstudyenrolled23neonatesborntoHIV-1-infectedmothers(EU)and43borntoHIV-1-uninfectedmothers (UN) (Table 1). All infected mothers receivedintravenous zidovudine (2 mg/kg weight, administered 1 hbefore c-section, and a zidovudine infusion at 1 mg/kg/hduring labor). Twenty-two of these 23 mothers receivedantiretroviral therapy during pregnancy, mainly zidovudine(600 mg/day) plus lamivudine (300 mg/day), and indinavir(800mg/every8h,tid)ornelfinavir(750mg,tid)ornevirapine(400 mg/day), according to institutional criteria. The infantswerenotbreastfedandweregivenHIV-1infectionprophylaxiswithoralAZTfor6weeks[19].Atthetimeofdelivery,40mlof CB was collected. A sample of peripheral blood (PB) wasobtained during the first 24 h after delivery, and at 1 and6 months of age to determine whether newborns wereperinatally infected by RNA virus load determination (RT-PCR  — Cobas, Roche, Indianapolis, IN). At 18 months, an additionalsamplewastakentodetermineanti-HIV-1antibodiesbyELISA.In addition, we included a cohort of babies born to HIV-1-infected and -uninfected mothers ( n =10 in each group) atdifferent ranges of age (3 to 6, 6 to 9, and 9 to 12 months), toevaluate DC number and phenotype. Four of the EU neonatesanalyzed at birth were also included in the follow-up. PBsamples from 10 healthy HIV-1-uninfected adults were used ascontrol (median age: 23 years, range: 19 – 43). Six out of these10 adult controls were nonpregnant women that were alsoincluded in the control group for pregnant women, HIV-infected or not. The remaining four individuals weremales, tomatch the male to female ratio seen in neonates.Institutionally approved informed consents were signed,according to Colombian government resolution 00843 of 1993legislation. Reagents Fluorochrome-labeled mAb against the molecules CD11c,CD123, HLA-DR, CD80, CD86, lineage markers (Lin-1, anti-CD3, CD14, CD16, CD19, CD20 and CD56), CD34 and thecorresponding isotype control antibodies was from BectonDickinson-Pharmingen (San-Jose, CA). The anti B7-H1 mAbwas from eBiosciences (San Diego, CA), while the anti-BDCA- Table 1  Characteristics of mothers and infants studiedMothers Newborns/InfantsAge Gestational(age in weeks)Viral load(copies/ml)CD4 counts(cell/ μ l)HIV-1 status ( ⁎ )Median (range) Median (range) Median (range) Median (range)HIV+ group ( n =23) 26 (18 – 42) 37.35 (34 – 39) 1370 (400 – 300,000) 480 (76 – 990) NegativeHIV −  controls ( n =43) 20 (18 – 40) 39.05 (37 – 41.2) NA NA Negative Results are expressed in median (range). All HIV-infected mothers were given AZT prophylaxis. NA: not applicable. ⁎ HIV-1 negative status was determined by viral load analysis at birth, 1 and 6 months and an ELISA test at 18 months of age. 244 P.A. Velilla et al.  Author's personal copy 2 and the Fc γ -receptor blocking reagent were from MiltenyiBiotec (Bergisch Gladbach, Germany).The C-Class CpG ODN 2395 (sequence 5 ′ -T CG T CG TTTT- CG G CGCGCG C CG -3 ′ ) was kindly provided by Coley Pharma-ceutical Group (Wellesley, MA). Culture of mononuclear cells Mononuclear cells (MCs) were isolated from PB or CB bydensity gradient; all samples were processed within 2 h of sample collection. MCs (1×10 6 /ml) were suspended inculture media (RPMI 1640, 10% heat-inactivated fetal bovineserum, 100 U/ml penicillin, 100  μ g/ml streptomycin and2 mM of   L -glutamine) and stimulated or not with 4  μ g/ml of C-Class CpG ODN 2395, or 1  μ g/ml of LPS from  Escherichiacoli  (serotype 026:B6, Sigma), or 1:400 dilution of a viableform of influenza virus (A/Bangkok, RX73 H3N2, Bioqual,Rockville, MD). Culture supernatants were collected after24 h and stored at  − 70 °C until cytokine determination. Flow cytometry FrequencyofmDCs(definedasLin − /CD34 − /CD11c+/HLA-DR+)and pDCs (Lin − /CD34 − /CD123+/HLA − DR+ or Lin − /BDCA 2+)and their expression of CD80, CD86 or B7-H1 were determinedby flow cytometry. In previous studies, we compared the pDCfrequencyusingbothmethods(Lin − /CD34 − /CD123+/HLA − DR+vs. Lin − /BDCA-2+), and similar results were obtained. Uncul-tured or cultured MCs were washed with cold PBS; nonspecificmAb binding was controlled by blocking Fc γ  receptors. MCswerethenresuspendedincytometrybuffer(PBS,0.5%BSAand0.1% NaN 3 ): 1×10 6 cells were incubated with the requiredmAbs for 20 min at RT in the dark.Due to the low DC frequency, 2.5 to 5×10 5 MCs wereanalyzed. Flow cytometry was performed using the BectonDickinson FACSORTand analyzed with the CellQuest software. Cytokine determination An ELISA kit was used to measure IFN- α , following themanufacturer ’ s instructions (PBL Biomedical Laboratories,Piscataway, NJ; detection limit  N 3.5 pg/ml). Statistical analysis Results are presented as medians and 25 and 75 interquartileranges. Statistical comparisons amonggroups wereperformedusing the Kruskal – Wallis test, with a confidence level of 95%.Statgraphics Plus, release 4, 1999 (Statgraphics Corp., Rock-ville, MD) and GraphPad Prism version 4.0 (GraphPad, CA)software were used for the analysis. The statistical signifi-cance was tested at  p b 0.05 as critical value using theStudent – Dunn ’ s post-test to compare medians among groups. Results Frequency of dendritic cells Circulating DC frequency was determined at birth (in CBMCs)andduring the first year of life (inPBMCs) in neonates born toHIV-infected mothers (EU) and HIV-uninfected mothers (UN).The  in utero  exposure to HIV was confirmed in additionalstudies in which CB CD4 + and CD8 + Tcells from 4 of these EUwere shown to proliferate and/or produce cytokines inresponse to a Gag peptide pool (results not shown). As shownin Fig. 1A, EU exhibited a 2-fold lower mDC frequency thanadult controls, although the difference was not significantlydifferent (0.24% vs. 0.52%,  p =0.11). A more pronouncedreduction in the percentage of circulating mDCs compared toadults was observed in UN (0.14% vs. 0.52%,  p b 0.01), thisfrequency being also significantly lower than that of EU(0.14% vs. 0.24%,  p b 0.05). When analyzed at different ageranges, the infants born to EU exhibit lower mDC frequencythan adults, similar to that observed at birth, although thedifference was not significant. The lower mDC frequencyfound at birth for UN also persisted over time whencompared to adult controls, although the difference wassignificant only at 6 – 9 and 9 – 12 months [median (range) of 0.19% (0.08 – 0.29) and 0.19% (0.11 – 0.31), respectively,compared with 0.52% in adults]. mDC frequency at differentage ranges was similar between both groups of children.At birth, EU and UN exhibited similar pDC frequency(0.17% vs. 0.11%, respectively,  p N 0.05; Fig. 1B), which waslower than in adults (0.34%), although the difference wassignificant only for UN compared to adult controls (  p b 0.01).pDC frequency at different age ranges was similar in bothgroups of children, and remained lower than in adults,although the differences did not reach statistical differenceexcept at 9 – 12 months for UN (0.34% vs. 0.13%,  p N 0.05). Nosignificant difference was found in the mDC/pDC ratiobetween EU and UN [1.3 (range: 0.3 – 10) vs. 1.1 (range0.07 – 8.5),  p N 0.216; data not shown].The evaluation of DC subpopulations in the mothersat delivery showed similar results to that observed intheir children. mDC and pDC frequencies were lower inboth HIV-1-infected and -uninfected mothers than innonpregnant women (mDCs: 0.28%, 0.14% and 0.53%,respectively; pDCs: 0.13%, 0.08% and 0.34%, respectively),but a significant difference was present only betweenHIV-1-uninfected mothers and nonpregnant women (forboth DCs,  p b 0.01). In contrast to the reported decreasedDC frequency in chronically HIV-1-infected nonpregnantadult women [20], the frequency of mDCs and pDCs wassignificantly higher in our HIV-1-infected mothers com-pared to uninfected mothers (both  p b 0.05). This discre-pancy could arise from the fact that all but one of theHIV-infected mothers included in this study were asympto-matic and on stable HAART at the moment of sampling.mDC and pDC frequencies in HIV-1-infected motherscorrelated positively with those observed in their neonates( r  =0.5,  p b 0.02 and  r  =0.56,  p b 0.006, respectively, Fig.2), whereas the same level of correlation did not existbetween uninfected mothers and their neonates ( r  =0.014,  p =0.93 and  r  = − 0.11,  p =0.52, respectively; data notshown). Expression of accessory molecules We determined the expression of the molecules CD80, CD86and B7-H1 on resting unstimulated CB mDCs and pDCs. Asshown in Fig. 3, mDCs and pDCs from EU and UN exhibited asimilar expression of both CD80 and CD86, and their levelswerenot significantly different from those observed in adults.245Intrauterine HIV exposure of dendritic cells
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